This invention relates generally to the analysis of samples such as biological tissue samples that are chemically stained for protein based markers, and more specifically to the use of a linking system and method for linking sets of images of the samples in such analysis.
Visual analysis of biological tissue samples often involves slicing the biological tissue samples into thin cross sections, often referred to as serial sections, to visualize structures of interest within the biological tissue sample. Serial sections are typically mounted on glass or plastic microscope slides. Visual analysis of mounted serial sections is often carried out by the naked eye (grossly) or by microscopy. In a typical slicing process, a tissue sample is dehydrated and embedded in paraffin to lend rigidity to the sample during slicing and mounting on a slide. Tissue samples are typically sliced into serial sections that are about 4–9 micrometers (μm) thick, however, other useful thicknesses are sliced. Once sliced, the serial sections are typically floated in water onto the microscope slides and moved into an appropriate location by a technician who physically manipulates the serial sections using, for example, a pair of tweezers or artist's brush. Being relatively thin, the serial sections are relatively delicate and when placed on microscope slides tend to deform by stretching, shrinking, being compressed, folding or a combination thereof Moreover, the serial sections also tend to be placed on the microscope slides in rotated positions relative to one another. Such deformations and relative rotations often add to the difficulty in cross comparing serial sections.
Serial sections of a tissue sample are typically cross-compared by histologists and pathologists, as well as others, to identify and locate the same tissue structure through the serial sections. For example, pathologists often cross compare serial sections that have been variously stained to aid in identifying and locating tissue structures of interest, such as groups of cancer cells or pre-cancerous cells. Stains of use have different affinities for different tissue structures and tend to color more intensely structures for which the stains have relatively high affinity. For example, a first serial section of a tissue sample is often stained with haematoxylin and eosin, referred to as H&E staining. Haematoxylin has a relatively high affinity for nuclei, while eosin has a relatively high affinity for cytoplasm. H&E stained tissue gives the pathologist important morphological and positional information about tissue of interest. For example, typical H&E staining colors nuclei blue-black, cytoplasm varying shades of pink, muscle fibers deep pinky red, fibrin deep pink, and red blood cells orange/red. The pathologist uses positional (e.g., color) information derived from the H&E stained tissue to estimate the location of corresponding tissue regions on successive serial sections of the tissue that are typically immunohistochemically stained. The successive serial sections may be immunohistochemically stained, for example, with HER-2/neu protein (a membrane-specific marker), Ki67 protein (a nuclei-specific marker), or other known stains. The use of such stains is well known in the art and will not be discussed in further detail.
Positional information derived from H&E stained serial sections is often crudely used to locate corresponding tissue on immunohistochemically stained serial sections. Pathologists commonly hold two or more slides up to a light and grossly attempt to judge the relative locations of structures of interest. As corresponding tissues may be distorted compared to the H&E section, and/or in a different location or orientation, position estimates may be many millimeters off leading to poor and/or lengthy-repetitious analysis.
Poor and lengthy analysis arise not only in naked eye analysis of serial sections but also in computer-aided analysis of serial sections. Images of serial sections are often digitized and stored in a computer for computer-aided analysis. Present computer-aided analysis techniques do not correct for distortions and relative rotations of serial sections captured in digital images of these sections. As a result of the distortion and relative rotations of a set of serial images captured in digitized images, using location information derived from one serial section image to locate structures in another serial section image using computer-aided techniques is a laborious process fraught with misidentification and lengthy, repetitious analysis.
Accordingly, what is needed in the fields of pathology, histology, morphology, and others are new and useful methods and tools to simplify and automate cross comparisons of serial sections. Also needed are new and useful methods and tools that provide improved positional accuracy during cross comparison of serial section images by correcting for serial section deformations and relative rotations that often arise during serial section slicing and mounting.